Cytomegalovirus drug resistance mutations in transplant recipients with suspected resistance

In this study, we developed a genotypic method of amplification through PCR and Sanger sequencing to analyse ARM in the UL54, UL56 and UL97 genes in clinical samples from 96 transplant recipients with suspected resistance to antivirals. To date, this is the study with the highest number of patients conducted in Spain. Moreover, according to a recent review of Chou S [13], we discovered a novel ARM “L397I” in UL97. Additionally, other three ARMs, such as L397R and H411L in UL97 and L516R in UL54, which were previously described as selected under drug in vitro, we detected them directly in clinical samples [13]. Interestingly, mutation at 397 position of UL97 confers resistance to maribavir despite this drug was not used in any patient. In this strain, high level GCV resistance ARM M460I (5–20 fold increase in ganciclovir 50% inhibitory concentrations) was also found, which suggests that GCV therapy could previously selected low level GCV resistance ARMs (< 2,2 Fold-increase in ganciclovir 50% inhibitory concentrations) producing cross-resistance to maribavir [13].

ARM was found in 18/67 (26.87%) patients regarding UL97, whereas 5.97% developed combined resistance to UL97 and UL54, and 4.54% to UL54 only. This rate was close to the 27% detected in SOT patients through Sanger sequencing in a previous study conducted in Barcelona [11]. Most ARMs were found in SOT patients, mainly in kidney and lung transplant recipients as described elsewhere [14, 15].

Most ARM was detected only in UL97 (14/21, 66.66%), indicating that the use of classical antivirals such as CDV and FOS, whose action mechanisms do not depend on UL97 kinase, is a reliable therapeutic option despite their wide use in transplant patients as alternative drugs. There was involvement of both UL97 and UL54 in 19.04% (4/21) of patients with ARM. Surprisingly, in three patients ARM was only found in UL54; this fact may be explained by the fact that some ARM in UL97 may have reverted to wild-type after switching therapy to FOS or CDV. In this sense, previous experiments have shown that, fortunately, the most common ARM found, L595S/W, reverts after a while, provided that the selective pressure of GCV is removed [16], suggesting a certain disadvantage of this ARM compared to susceptible wild-type. Of note is the high proportion of patients with treatment failure unrelated to ARM: 72,72% (48/66) and 89,23% (58/65) regarding UL97 and UL54, respectively. Unknown factors probably related to the patient’s condition and/or virus virulence may be also responsible for most refractory CMV infections. Therefore, the absence of response to treatment is not decisive to establish a case of antiviral resistance, and confirmation with genotypic methods [11, 13] is required at any rate [17]. Only two HSCT patients had not refractory CMV infections, which is in agreement with previous studies indicating that resistant CMV infections remain a rare complication in HSCT recipients, whereas refractory infections are more commonly found [18].

In this study, we searched for consensus ARM related to the lack of effectiveness of the main antivirals used against CMV (GCV, FOS, CDV, VGCV and LET) (Table 1). The presence of each of the mutations can affect a single drug or several ones simultaneously. Among the mutations found in the UL97 gene, H520Q/E and C603W/R/S were previously associated with high rates of resistance to GCV. However, the role of others, such as D605E, is controversial and, depending on the study, may be regarded as a resistance mutation or a variant of the natural sequence [19]. Recent recombinant phenotypic experiments indicated that this mutation did not confer resistance to GCV [13]. Therefore, we did not consider D605E, found in three patients, as an ARM.

Concerning resistance to LET, previously described ARMs were related to mutations located between amino acids 230 and 370 of UL56 [10, 18]. In vitro and clinical studies showed that ARM developed faster than in UL97 and UL54, which is a reason for increasing concern among clinicians and virologists. Regarding UL56, since two naturally occurring sequence polymorphisms (L241P and R369S) were described to confer 160-fold and 38-fold reduced susceptibility to LET [20], respectively, we decided to study this gene despite only one patient with suspected resistance was treated with LET and, even with treatment failure, no ARM was found in UL56. Although the main target of ARM to LET has been found in UL56, other ARMs in UL51 and UL89 could not be ruled out. Seven patients with ARMs in UL54 were found, four of them with combined ARMs in UL97, which suggests that most of the ARMs were accumulated in UL97 kinase when GCV or a closely related antiviral as VGCV was used. This finding is in agreement with previous studies, in which more than 90% of ARMs occurred in the UL97 gene, specifically between codons 460–520 and 590–607 [3, 6, 13, 15]. Other antivirals, such as FOS and CDV could be used instead in these cases, which highlights the importance of genotypic determination of ARMs for a right therapeutic choice. ARM was also found in UL54 DNA polymerase being T503I the most common (3/7 patients) which has been described as conferring resistance to GCV and CDV as well as A987G (2/7 patients). One patient developed multiple ARMs in UL54, one of which (A834P) is related to the appearance of resistance to FOS [19, 21].

In addition to the above-mentioned ARMs, other mutations compared to reference wild-type strains were found because of a certain polymorphism in UL54. The frequency of some of them is high, as in the case of S655L (51.14%) and F669L (42.86%) located at UL54. However, their consideration as candidate ARMs requires further recombinant phenotypic or marker transfer studies. It should be noted that the occurrence of multiple ARMs, which markedly increases antiviral resistance, thus complicating prognosis and treatment management [22, 23], was a common event: (8/21) of patients with ARMs.

In the search of ARMs in cohorts of patients with suspected resistance to antivirals, efforts have been made in many laboratories worldwide to develop NGS-based methods due to their ability to multiplex large numbers of samples. However, in our experience, for routine virological screening with few patients, NGS assays are still quite costly and time-consuming compared to PCR and Sanger sequencing. The main advantage of NGS was that ARMs may be characterised in samples with lower viral load [11] or when minor resistant subpopulations exist.

Despite limitations, the findings of this work contribute to reinforce the observation of the presence of mutations associated with drug resistance previously described, while making a case for the discussion on the involvement of new ones in the emergence of antiviral resistance. It is also shown that drug resistance is an important feature of CMV pathogenesis in transplant recipients that may threaten transplant outcomes, while the value of genotypic testing to identify potential antiviral resistance mutations is highlighted, which in turn could contribute to a better virological diagnosis and clinical performance.